Method of Observing the Emission of Light from a Sample by Dynamic Optical Microscopy

ABSTRACT

Method for observing an emission of light ( 14, 15 ) from a sample ( 10 ) in a medium ( 11 ) of refractive index n L  disposed against a surface ( 20   a ) of a transparent support ( 20 ) of refractive index n s , greater than n L , the emission of light comprising luminous components oriented toward the support and forming an angle θ with a direction ( 20   b ) perpendicular to the surface ( 20   a ), said components including supercritical luminous components and critical or subcritical luminous components, the method implementing an observation device ( 100 ) capable of collecting at least part of the emission of light, of applying filters ( 170 ) to the luminous signal collected; and of transforming the filtered luminous signal into an image zone of the sample ( 6   a,    6   b ); the method being characterized in that:
         A modulation of the filtered luminous signal is carried out, in which luminous components arising from the critical or subcritical luminous components of the emission of light are allowed to pass through so as to obtain image zones ( 6   a,    6   b ) of one and the same region of interest of the sample, the modulation pertaining to all or some of the luminous components of the collected luminous signal which arise from the supercritical luminous components of the emission of light; and   At least one useful image zone ( 6   c ) of the sample is produced by combining image zones ( 6   a,    6   b ), the combination evidencing differences between the image zones ( 6   a,    6   b ) related to the modulation.

The present invention relates to a method of observation of the emissionof light from a sample by dynamic optical microscopy.

The light from the sample may result from diffusion or fluorescence.Fluorescence microscopy is a technique that takes advantage of thephenomenon of fluorescence in order to observe various compounds.Fluorescence is the property possessed by certain bodies to emitfluorescent light by themselves.

The fluorescence of an observed compound can be primary, if the compoundis fluorescent itself (e.g., chlorophyll, oil) or secondary, if theobserved compound is marked with a fluorescent substance known as afluorochrome or fluorescent marker.

In particular in cell biology, a large number of molecular eventsoccurring at the cell surface are studied by fluorescence microscopy,such as cell adhesion, the binding of hormones to receptors in theplasma membrane, the secretion of neurotransmitters as well as membranedynamics (endocytosis, exocytosis).

A fluorescence microscopy device usually comprises a light source forexcitation, means for separating the excitation photons from theemission photons, a lens system for capturing the photons and, ingeneral, imaging means.

Fluorescence techniques can be used with different types of microscopes,notably:

-   -   A conventional optical microscope where the excitation light may        pass through the sample or the lens. In the latter case, this is        epifluorescence microscopy;    -   A confocal microscope, for example by laser scanning that in        particular enables three-dimensional images of the sample;    -   A total internal reflection fluorescence microscope (usually        called TIRF), which uses an evanescent wave to excite the        fluorescence that is of only a very shallow depth, immediately        adjacent to the interface of the sample substrate (usually        glass) and of the liquid medium (usually water), in which the        sample is disposed. Lighting is performed by a laser beam        incident at a supercritical angle to create an evanescent wave        (exponentially decreasing orthogonally to the interface).

TIRF microscopy, although currently booming and allowing preciseobservations, has some disadvantages. Indeed, the use of an adaptedlaser source is costly and the excitation field thus generated may notbe homogeneous (due to interference from the coherence of the beam). Inaddition, lighting by the lens does not allow a homogeneous excitationand the resulting depth of penetration is not constant across the fieldto be observed. Moreover, there are containment losses of the excitationfield related to the intrinsic light scattering by the cells.

FR-A-2943428 discloses a method of observation by fluorescencemicroscopy of a sample comprising fluorescent components in a liquidmedium of refractive index n_(L) arranged on a transparent support ofrefractive index n_(s), which is greater than n_(L) and less than orequal to 1.55, and an observation device comprising a full-fieldimmersion lens, whose numerical angular aperture, ON, is greater than orequal to 1.33 and less than or equal to n_(s), and a set of lenses forforming an image in at least one image plane, and which furthercomprises a mask arranged in the rear focal plane of the immersion lensor a conjugate plane of said rear focal plane, so as to obscure thefluorescence emission components of the sample in the angular directionsin which the angle θ is less than or equal to a critical angle θ_(c),with θ_(c)=arcsin (n_(L)/n_(s)), and the angle θ defined as the angle ofan angular direction of the fluorescence emission relative to theperpendicular direction of the support surface on which the sample to beobserved is arranged.

This observation method permits obtaining high-quality fluorescenceimages with a low-cost device or improving the quality of imagesobtained by TIRF microscopy.

The device and the method described above lead to very satisfactoryimages with good resolution. Nevertheless, in certain circumstances, onemay wish to improve the resolution.

The present invention aims to provide an alternative method permittingin particular to improve substantially the image resolution at the costof some changes.

The solution of the invention is a method for observing an emission oflight from a sample in a medium of refractive index n_(L), the samplebeing placed against a surface of a transparent support of refractiveindex n_(s), which is greater than n_(L), wherein the light emissioncomprises luminous components of a given amplitude and phase, orientedtoward the support and forming an angle θ with a direction perpendicularto the surface, including, on the one hand, supercritical luminouscomponents, for which the angle θ is strictly greater than a criticalangle θ_(c)=arcsin (n_(L)/n_(s)), and, on the other hand, critical orsubcritical luminous components, for which the angle θ is less than orequal to the critical angle θ_(c), whereby the method implements anobservation device capable of:

Capturing at least part of the light emitted from a region of interestof the sample and obtaining a captured luminous signal that comprisesluminous components emitted from the supercritical luminous componentsof the light emission;

Applying filters to the captured luminous signal in order to selectivelydecrease the amplitude and/or change the phase of certain luminouscomponents of the captured luminous signal in order to obtain a filteredluminous signal; and

Transforming the filtered luminous signal into an image zone of theregion of interest of the sample;

the method being characterized in that:

A modulation of the filtered luminous signal is realized, whereinluminous components arising from the critical or subcritical luminouscomponents of the light emission are allowed to pass through in order toobtain image zones from one and the same region of interest of thesample, the modulation pertaining to all or some of the capturedluminous signal's luminous components emitted from the supercriticalluminous components of the light emission; and

at least one useful image zone of the sample is produced by combiningimage zones, wherein the combination evidences differences between theimage zones pertaining to the modulation.

The light emitted by the sample may result from fluorescence (after anappropriate excitation) or from diffusion. A portion of this light iscaptured by an observation device, then filtered and converted into animage zone.

The region of interest of the sample is the part of the sample that isto be observed. It can be extended or isolated. In the latter case, itis possible to reconstruct a larger image by scanning the sample. Animage zone is an image of a region of interest of the sample. It maythus be a full image of the sample, an image of a portion of the sample,or even an image of a point of the sample. The modulation applies toimage zones issued from one and the same region of interest of thesample.

With the device according to the invention, it is possible to obtainmicroscopy images with an improved resolution, allowing for example toobtain valuable information for the study of biological materials.

The resolution improvement is obtained not by working directly on aluminous signal purged of all or part of the components arising from thelight emitted by the sample at critical or subcritical angles, that isto say a signal rich in components arising from the light emitted by thesample at supercritical angles (the method described in the documentFR-A-2943428), but rather by working indirectly, by a modulation of thesignal applied to the components arising from the light emitted by thesample at supercritical angles. The method is indirect because itrequires taking several image zones of the sample and combining them toobtain a useful image zone that evidences the differences between theimage zones created by the modulation of the luminous signal at theorigin of the image zones.

The modulation being applied to all or some of the luminous componentsof the captured luminous signal arising from the supercritical luminouscomponents of the light emission, the combination enhances thesesupercritical luminous components in the useful image zone. By contrast,the components of the captured luminous signal arising from the lightemitted by the sample at critical or subcritical angles not beingaltered by the modulation, the combination reduces these critical orsubcritical components in the useful image zone. The combination can beseen as a demodulation. It shows in the useful image zone thecontribution of the modulated supercritical components.

The modulation can affect the amplitude and/or the phase of the luminouscomponents of the captured luminous signal. In other words, the filtersused alter the amplitude and/or phase of the light waves. This is alsothe meaning of the verb “to act,” when it is used relating to thefilters in this application. To act differently, for two filters, means“to achieve a modulation.” Changing the amplitude can be a more or lessstrong attenuation going up to full obscurement. Some of the filtersused (but not all) can be neutral and not act; in this case, theresulting luminous signal is called a “filtered signal” anyway, even ifit is identical to the captured luminous signal. “Passing” luminouscomponents means that after filtering, the components remain, eventhough in an attenuated form (reduced amplitude) or with a modifiedphase.

The image zones, just like all images formed using a sensor such as acamera, contain information on the luminous intensity related to thesquare of the module of the filtered luminous signal.

Unexpectedly, the obtained resolution of the useful image zone can beimproved by a factor of about 20 to 25%, especially if the modulation isapplied to all the luminous components of the captured signal arisingfrom the supercritical luminous components of the light emission (seethe explanation of FIG. 7 below) with respect to an image zone obtainedby the method of the document FR-A-2943428.

Taking image zones, in order to obtain a useful image zone, can be donesimultaneously, for example by splitting the captured luminous signaland applying a filter to each split signal. Taking image zones can alsobe done successively. A luminous signal is then captured and filters aresuccessively applied to it thus realizing the modulation. An image zoneis stored for each filtered luminous signal obtained.

The method provides at least one useful image zone, but it can beapplied as often as necessary, at a rate permitted by the observationdevice and the filters used, so as to obtain useful image zones at giventime intervals. If the image zones correspond to points of the sample, aspatial scanning of the sample can be achieved.

The invention allows obtaining image zones with high sensitivity,especially zones localized at the interface between the support ofrefractive index n_(s) and the medium of refractive index n_(L). One canthus visualize events occurring near this interface to a certain depth.For an interface of glass/water (about n_(L)=1.33 and n_(s)=1.51), thedepth may be about 1/6 of the wavelength, or less if only collecting thesteeper luminous components (the most supercritical ones) of the emittedlight, of the order from 50 nm (nanometers) to 100 nm for wavelengths inthe visible zone.

Supports commonly used in microscopy, in particular fluorescence, aremade of glass, and it is possible to choose conventional types of glass,of a refractive index less than or equal to 1.55, the cost of which islow, or glass types of a higher index.

Fluorochrome behaves as an antenna capable of transmitting a signal.This transmitter has in its immediate environment (a few tens ofnanometers) electromagnetic components, which are evanescent when placedin a homogeneous medium. These components may become propagative whenthe fluorochrome is positioned near an interface. They then propagate atangles that are larger than the critical angle in the medium with thehighest refractive index.

FIG. 5a shows the components of the emission of fluorescence of atransmitter 12 located at the interface between a support 20 and aliquid medium 11.

The fluorescence emission of the transmitter 12 comprises a component 14emitted for θ between 0 and θ_(c), and a component 15 emitted for θgreater than θ_(c), called “no-light” or “supercritical light” andcorresponding to evanescent components in the liquid medium 11 and thatbecome propagative in the transparent support 20.

For example, for a glass/water interface, we find that the supercriticallight can be up to about a third of the intensity of the total lightemitted.

The value of the critical angle is given by the Snell-Descartes laws ofrefraction, where θ_(c)=arcsin (n_(L)/n_(s)). In the case where theliquid is water (n_(L)=1.33) and the support is standard glass(n_(s)=1.52), θ_(c) is 61°.

FIG. 5b shows the components of the emission of fluorescence of atransmitter 12 located at a greater distance of about 100 nm.

It has been found that the emitted light comprises the same component14, issued with θ between 0 and θ_(c), but no longer comprisescomponents emitted at an angle greater than θ_(c), corresponding to thesupercritical light.

The method according to the invention allows to retrieve the informationcontained in the supercritical light and thus to produce useful imagezones with information on structures and their possible evolution in the100 nm depth from the liquid medium/support interface.

The numerical aperture, ON, of a lens is defined by ON=n. sin(α_(max)),where n is the index of the operating environment of the lens andα_(max) is the maximum collection angle of the lens.

Current commercial lenses with high numerical apertures with oilimmersion (n=1.51) have been developed. They have ON, for example, of1.45 and 1.49. The maximum captured angles are greater than the criticalangle θ_(c). Thus, these lenses can collect most part of thesupercritical light. Preferably, these lenses are corrected forspherical and chromatic aberrations and thus permit obtaining anexcellent-quality full-field image.

According to particular embodiments of the invention, the invention mayimplement one or more of the following characteristics:

The luminous components of the captured luminous signal that are subjectto said modulation are emitted from supercritical luminous components ofthe light emission for which the angle θ is within a predetermined rangein accordance with a range of depths to be explored in the sample.

The image zones obtained by using the observation device and producing,by combination, the useful image zone of the sample are successivelyobtained by successively applying filters to the captured luminoussignal.

The method may comprise the following steps:

a) taking a plurality of image zones in the same region of interest ofthe sample using the observation device and a plurality of filters, eachfilter used for taking an image zone of said plurality of image zones,the plurality of filters being such that:

-   -   A filter of said plurality of filters allows the passing through        of, in the filtered luminous signal, luminous components arising        from the supercritical luminous components of the light        emission;    -   The filters of said plurality of filters all allow the passing        through of luminous components of the captured luminous signal        emitted from critical or subcritical components of the light        emission and act in a substantially identical fashion between        them on the luminous components of the captured luminous signal        arising from the critical or subcritical luminous components of        the light emission, and    -   There are at least two filters of said plurality that act in a        substantially different fashion between them on the amplitude or        the phase of at least part of the luminous components of the        captured luminous signal arising from supercritical luminous        components of the light emission; and        b) producing a useful image zone of the sample by a calculation        combining the plurality of image zones taken in step a) in order        to evidence differences between the image zones of the plurality        of image zones of the sample.

The method may comprise the following steps:

a) taking at least two image zones of one and the same region ofinterest of the sample using the observation device and two filters,each filter used for taking one of the two image zones, the two filtersbeing such that:

-   -   One of the two filters lets luminous components arising from the        supercritical luminous components of the light emission pass        through in the filtered luminous signal; and    -   The other filter acts in a substantially identical fashion to        said one of the two filters on the luminous components of the        captured luminous signal arising from the critical or        subcritical luminous components of the light emission and it        decreases, to a substantially greater degree than said one of        two filters, the amplitude of at least part of the luminous        components of the captured light signal arising from the        supercritical luminous components of the light emission; and        b) producing a useful image zone of the sample by a calculation        combining the two image zones of the sample taken in step a),        the calculation comprising an algebraic difference between the        two image zones of the sample.

The filters implemented also partially reduce the amplitude of all orsome of the luminous components of the captured luminous signal arisingfrom the critical and subcritical luminous components of the lightemission.

Luminous components of the captured luminous signal arising from theluminous components of the light emission at the same angle θ areprocessed in a substantially identically fashion by a same filter forthe reduction of the amplitude or the change of the phase.

The sample displaying a phenomenon to be observed with a givencharacteristic time, the image zones are successively taken at timeintervals of less than or equal to half the characteristic time.

One of the image zones of the sample is obtained using a neutral filterthat allows to pass through, in the filtered luminous signal and withoutany decrease of amplitude, all the luminous components of the capturedluminous signal arising from the supercritical luminous components ofthe light emission; and another image zone of the sample is obtainedwith a total filter, which cancels in the filtered luminous signal allthe luminous components arising from the supercritical luminouscomponents of the light emission.

The observation device comprises a full-field immersion lens and thefilters are located in a rear focal plane of the immersion lens and/orin a conjugate plane of said rear focal plane.

The filters comprise a diaphragm which, in an open position, allows thepassing through of luminous components from the captured luminous signalarising from the supercritical luminous components of the light emissionand that, depending on the degree of closure, allows the obscuring ofthe luminous components of the captured luminous signal arising from thesupercritical luminous components of the light emission, having an angleθ greater than a limit value related to said degree of closure.

The sample to be observed is biological in nature.

According to a particular embodiment, the modulation does not affect allthe luminous components of the captured luminous signal arising from thesupercritical luminous components of the light emission. It may affectonly those for which the angle θ is within a predetermined set, forexample, the interval [θ_(a), θ_(b)], θ_(a) and θ_(b) being bothstrictly greater than θ_(c). This allows the exploring of the range ofcorresponding depths in the sample (see explanation for FIG. 1).

In another embodiment, the method includes a step a) of taking aplurality of image zones corresponding to modulated luminous signals.For the modulation, it is necessary that at least one of the filtersused allows the passing through of luminous components from thesupercritical part of the light emission. It is also necessary that theother filters act differently from this filter on all or part of thesupercritical components of the captured luminous signal. “Differently”can mean that the filters in question attenuate more, or less, theamplitude of the luminous components involved. The difference may alsorelate to the phase. The actions on the amplitude and phase can becombined.

On the other hand, the filters must act in a substantially identicalmanner on the luminous components arising from the critical orsubcritical part of the light emission. The luminous components arisingfrom the critical or subcritical part of the light emission can bechanged by the filters used for obtaining the image zones, but thischange should be substantially the same for all the combined image zonesin order to get the useful image zone. “Substantially the same” meansthat in a particular embodiment, there is no difference, but, accordingto another embodiment, there may be minor differences from the desiredmodulation (the modulation of the luminous components arising from thesupercritical luminous components of the light emission). Preferably,luminous components arising from the critical or subcritical luminouscomponents of the light emission will be left in the filtered luminoussignal.

The method comprises a step b), where image zones taken in step a) arecombined. The combinations are made by a calculation based on the lightintensity. This calculation is based on the filters used and is used toevidence the differences between the image zones caused by themodulation.

The advantage of taking a plurality of image zones is to reduce anybackground noise, for example by introducing averages in thecalculation.

For example, one can take three successive image zones of a same regionof interest of the sample. The first one is an image zone of the samplethat contains information related to all the luminous components arisingfrom the supercritical part of the captured signal, that is to say, thefilter used has not substantially modified these components. The secondone is an image zone that contains no information related to theluminous components from the supercritical part of the captured signal,that is to say, the filter used has obscured these components. The thirdimage zone is taken under conditions identical to the first image zone.A possible combination of these three successive image zones is tocompute the absolute value of the difference between, firstly, anaverage of the first and third image zones and, secondly, the secondimage zone. This can allow the reduction of noise in the useful imagezone (photo-bleaching phenomenon, changes in excitation intensity,movement of the sample).

The images contain information related to the (positive) intensity ofthe overall electric field. A difference between two pixels can thus bepositive or negative, which is why an absolute value is used to obtain apositive result that represents an intensity, namely that of the usefulimage zone.

According to another particular embodiment, in step a) two image zonesare taken of one and the same region of interest of the sample. Thefirst one is taken using a filter that allows the passing through ofluminous components from the supercritical part of the light emission.The second one is taken using a filter that acts differently from thefirst filter upon all or part of the supercritical components of thecaptured signal, and substantially identically upon the critical orsubcritical components of the captured signal. In step b), the two imagezones are combined by computing for each pixel the absolute value of thedifference between the first and the second image zones. This resultrepresents an intensity, namely that of the useful image zone. Theadvantage of this embodiment lies in the simplicity of the combinationof the image zones, that is to say, the simplicity and speed of thecalculations.

According to a particular embodiment, one can reduce in the two imagezones, in an identical fashion, part or all of the luminous componentsof the captured luminous signal arising from the critical or subcriticalluminous components of the light emission. This is important when thelight originating from the subcritical rays is very intense comparedwith that originating from the supercritical rays.

In general, the filters used respect the symmetry of revolution andtreat in the same way the luminous components of the captured luminoussignal arising from the luminous components of the light emissionforming the same angle θ. Indeed, a modulation on the azimuth of thecomponents of the light emitted does not present much interest. On thecontrary, not modulating the azimuth prevents the introduction ofastigmatism into the useful image zone.

If the sample changes over time (such as a cell membrane) with a givencharacteristic time, one takes successive image zones (ratherrepresentative of the sample in depth) with a period shorter than halfthe characteristic time (video frame rate) in order to monitor thesechanges. The method normally allows the obtaining of useful image zones(rather representative of the sample interface) at the same rate as thatof the successive image zones, i.e., at the video frame rate and not athalf of the video frame rate as in the method described in the documentFR-A-2943428.

The addition of a diaphragm in the rear focal plane of the lens and/orin a conjugate plane can be done easily and in particular can beimplemented with commercial microscopes. The result is a device whosecost of the improvement is very modest.

The function of the diaphragm is to obscure the luminous components ofthe captured luminous signal arising from the luminous components of thelight emission from the sample in angular directions θ greater than orequal to a certain angle depending on the aperture of the diaphragm.FIGS. 5a and 5b refer to the principles of fluorescence emission. Anadvantage of the method is that the subcritical information is alwaysavailable in the image zones used for the combination. This informationis often interesting, because it is connected to the innermost regionsof the sample.

Other features and advantages of the present invention will becomeapparent from the following description of non-limiting exemplaryembodiments, with reference to the accompanying drawings, wherein:

FIG. 1 shows a schematic view of a microscopy device allowing toimplement a method according to the invention;

FIG. 2 shows a variant according to the invention of the apparatus shownin FIG. 1, suitable for confocal microscopy;

FIG. 3 shows a variant according to the invention of the apparatus shownin FIG. 1, suitable for TIRF microscopy;

FIG. 4 shows an example of a filter for the implementation of a methodaccording to the invention;

FIGS. 5a and b show the fluorescence emission components according todifferent configurations and have been commented on above;

FIGS. 6a and 6b illustrate the image zones of a same cell respectivelywith and then without the luminous components of the captured luminoussignal from the critical or subcritical luminous components of the lightemission;

FIG. 6c shows a useful image zone of the same cell obtained by combiningthe image zones of FIGS. 6a and 6 b;

FIG. 7 is a graphical representation of the light intensity of the spotsobtained from a same test sample, firstly by the method described in thedocument FR-A-2943428 (obscuring of critical or subcritical components)and secondly by a method according to the invention (modulation appliedto the supercritical components, and then demodulation).

For reasons of clarity, the dimensions of the various elements shown inthese figures are not necessarily in proportion to their actualdimensions. In the figures, identical references correspond to identicalelements.

FIG. 1 shows a schematic view of a fluorescence microscopy device 100.It comprises an immersion lens 110, of which the ON is greater than orequal to 1.33. A glass support 20 is arranged above the immersion lens110. Oil is disposed between the immersion lens 110 and the glasssupport 20.

A sample 10 to be observed is arranged on the glass support 20. Thissample 10 comprises, for example, fluorescent elements dispersed inwater.

The rear focal plane of the lens is referenced with number 400.Excitation light is generated by a beam 200 from a light source, whichpasses through an excitation filter 210 and is reflected by a dichroicmirror 120 to illuminate the sample 10 after passing through thetransparent support 20. An example of the path of the incidentexcitation light is indicated by the arrows pointing to the top of thefigure. The incident excitation light may be partly reflected and isthen filtered by an emission filter 130 so that the image formed on animage plane comprises only the fluorescence light emitted by the sample10.

The fluorescent light emitted by the sample 10 passes through thetransparent support 20, the dichroic mirror 120, and the emission filter130.

According to the embodiment shown in FIG. 1, this light is reflected offa mirror 140 and the rest of the device works with the reflected light.

A lens 150, called tube lens, allows the focusing of the light on anintermediate image plane 410.

Then the light is parallelized by a lens 160 and focused by a lens 180onto the image plane 430, where the image is acquired by a suitabledevice, notably by a camera 300. The planes 430 and 410 are conjugateimage planes of the observation plane.

The lenses 160 and 180 are arranged so that a conjugate plane 420 of therear focal plane of the immersion lens 110 is located between the lenses160 and 180.

A variable-aperture diaphragm 170 is arranged in the rear focal plane ofthe immersion lens. This diaphragm acts as a filter for the luminouscomponents of the captured luminous signal. It can be in open positionand allow the passing through of all the luminous components of thecaptured luminous signal arising from the luminous components of thelight emission. It can be in a partially closed position and obscurepart of the luminous components of the captured luminous signal.

More specifically, the light rays that are emitted following a certainangle by the fluorescent emitters of the sample 10 located in theobservation plane intercept the rear focal plane 400 of the lens (or anyconjugate plane 420 of the plane 400) at a certain distance r(θ) fromthe center (defined by the optical axis) of this plane. r(θ) is anincreasing function of θ. For aplanatic lenses, for example, r(θ) isapproximately proportional to sin(θ). Thus, all the rays emitted at theangle θ (conically) describe a circle of a radius of r(θ) in the rearfocal plane.

When the diaphragm 170 is arranged in the rear focal plane 400 of thelens 110, the relationship between r(θ) and sin(θ) is:

r(θ)=n_(i)×f_(o)×sin(θ), where f_(o) is the focal distance of theimmersion lens 110 (usually of the order of a few millimeters) and n_(i)is the index of the immersion medium used for the lens (usually oil).

According to an embodiment, there is an immersion lens with amagnification G=100 and the focal distance of the tube lens 150 isf_(t)=200 mm.

We then have f_(o)=f_(t)/G=2 mm. In this configuration we get: r(θ_(s))=2.66 mm.

If the diaphragm 170 is arranged in the conjugate plane of the rearfocal plane, one should take into account the magnification factorrelated to the optical system. For example, in the plane 420, it isnecessary to introduce a multiplication factor G′=f160/f150 where f150is the focal distance of the tube lens 150 and f160 is the focaldistance of the lens 160.

The luminous components of the captured luminous signal arising from thecritical or subcritical luminous components of the light emissionintercept the rear focal plane following a closed centered disk of aradius of r(θ_(c)). The luminous components of the captured luminoussignal arising from the supercritical luminous components of the lightemission, where θ_(c)<e<θ_(max), form an open ring in the rear focalplane r(θ_(c))<r(θ)<r(θ_(max)). The implementation of a diaphragm 170centered on the optical axis and having an opening r(θ_(c)) thus allowsto obscure all supercritical luminous components.

The selection is thus made at the emission. As a result, the lightingsystem does not need to be changed from that of a standardepifluorescence observation device. It is thus possible to illuminatewith a source of non-coherent light, such as a standard white light,obtained in particular using a mercury lamp. This results in severaladvantages, such as the absence of significant additional cost (comparedto TIRFM microscopy technique, where a laser light is required) as wellas the possibility to obtain a homogeneous field (possibly allowingquantitative measurements).

According to one embodiment, actuation means of the diaphragm operate atthe video frame rate (typically in the magnitude of a few tens of Hertz)in order to pass alternatively from the open position to the closedposition with the speed of image acquisition. It is thus possible tohave information on the volume and the surface simultaneously.

This imaging method is particularly suitable for imaging biologicalsamples, in particular for the study of biological processes in livingcells, such as cell adhesion phenomena, endocytosis/exocytosis, . . . .

FIG. 2 shows a schematic view of a variant according to the invention ofthe device of FIG. 1 where the elements present before the image plane430 are identical in both embodiments. In the device of FIG. 2, apinhole-type mask 190 that comprises a hole 195 is arranged in the imageplane. A mono-detector 350 allows a point-by-point acquisition of thelight passing through the hole 195. It is thus possible to obtain aconfiguration that allows performing the confocal microscopy.

FIG. 3 shows a schematic view of a variant according to the invention ofthe device of FIG. 1 where the elements of the microscopy device aresimilar, but where the light source differs.

In the device of FIG. 3, the light 250 is coming from a laser and theillumination of the sample is produced by total internal reflection. Itis thus possible to obtain an improved TIRF type device.

Note that the rear focal plane of the commercial lenses is usuallylocated inside the lens and is therefore difficult to access. It is thusoften recommended to realize a system for imaging the rear focal planeto be able to insert the filter system 170 between the sensor and thelens.

According to one embodiment, an inverted fluorescence microscope of theTi type of Nikon is used that comprises a module (ref. TI-T-BHP,MEB55810) that enables imaging the rear focal plane and positioning anannular mask to enable the (external) phase contrast with largenumerical aperture lenses. It is possible to put into this type ofmodule a diaphragm 170 of the device according to the invention. Thesystem of centering and adjusting the position of the plane is quitesuitable for a diaphragm filtering supercritical angles. The systemcomprises a plurality of positions for different lenses.

FIG. 4 shows a schematic view of a diaphragm 170 to be arranged in therear focal plane 400 of the immersion lens 110 or in a conjugate plane420 of said focal plane. The diaphragm 170 comprises a peripheral area176 apt at obscuring light. This area 176 is either actually mobile (asin a camera), or the diaphragm is replaced by another, for example, byturning a motor-driven rotary filter wheel.

The diaphragm 170 may be an iris diaphragm, such as that sold byThorlabs. Its aperture is adjusted by moving mechanical moving parts(not shown).

Another possibility is to use a wheel with openings or semi-transparentmaterials distributed on sectors of the wheel and rotate it. In thiscase, the diaphragm can, for example, be achieved by a circular hole ofa suitable diameter in an opaque material. This allows the obtaining ofvery short transmission/shutter cycles, which, for example, keep pacewith the acquisition pace of images with a camera.

According to one example of an embodiment, a Nikon “Ti-U”® type invertedfluorescence microscope is used with a base of the binocular tube of thephase “TI-T-BPH”®, an oil-immersion ×100 lens with a numerical apertureof 1.49. A fluorescence filter cube, which contains a transmissionfilter, a dichroic plate, and an excitation filter, is used. The lightsource used is a fiber source of the commercial reference “NikonItensilight”® with a 130 W Hg lamp and a generator “C-HGFI”®. The cameraused is an EMCCD Andor Ixon+ camera, cooled to approximately −75°. Thediaphragm 170 used is the iris diaphragm produced by Thorlabs.

The diaphragm is then positioned in the Nikon MEB55810 module(“TI-T-BPH”) instead of the phase ring. The position of the diaphragm isadjusted by means of the Bertrand lens of the microscope and bydisplacement of the module by means of screws for centering and axialposition. The procedure followed is the same as the adjustment of thephase ring supplied by the manufacturer with the module.

Observations have been conducted on embryonic human kidney cells markedby Choleratoxin (which binds to glycolipids on the membrane and theconstituents of lipid rafts) coupled to Alexa 488 and excited by theNikon Intensilight Hg 130 W lamp (conventional lamp). The filter cubeused consists of an excitation filter with a bandwidth from 450 to 490nm, a dichroic mirror of 500 nm, and an emission filter with a bandwidthfrom 510 to 550 nm.

FIGS. 6a and 6b show two images obtained with an open diaphragm (6 a)and with a closed diaphragm to hide all the supercritical luminouscomponents (6 b). The pause time (T=300 ms) and the gain (G=0) areidentical for images 6 a and 6 b. The image 6 c is obtained as theabsolute value of the difference between the images 6 a and 6 b (that isto say, between the intensity of the signals associated with theimages).

It is noted that the two images 6 a and 6 b appear to be identical.However, image 6 c is well contrasted. One advantageously observesintensity variations that are associated with membrane phenomena thatare difficult to distinguish in the other two images, because they areembedded among other information coming from the inside of the cell.

It should be noted that these observations have been advantageously madewith a “classic” lamp and that it was not necessary to implement a laserto obtain them.

Measurements of the lateral resolution have been performed withfluorescent beads Fluosphere® (marketed by Invitrogen) of theexcitation/emission: 580/605 nm deposited by spin (“spin-coating”) on astandard glass slide (thickness 0.13-0.16 mm), and then immersed indistilled water.

FIG. 7 illustrates the profile of the fluorescence intensity of thesebeads (normalized signal intensity on the ordinate as a function oflateral displacement on the abscissa, expressed in microns). It shouldbe noted that the C2 profile, which corresponds to the useful image zoneobtained by the method according to the invention is narrower than theC1 profile, which corresponds to the image obtained by the methoddescribed in document FR-A-2943428. The corresponding improvement inresolution is 20-25%.

The invention is not limited to these types of embodiments and should beinterpreted in a non-limitative way and encompassing all equivalentembodiments.

1.-12. (canceled)
 13. An observation device suitable for observing alight emission from a sample in a medium with a refractive index n_(L),the sample being arranged on a surface of a transparent support ofrefractive index n_(s), which is greater than n_(L), the light emissioncomprising luminous components which each have an amplitude and a phase,and are oriented toward the support and forming an angle θ with adirection perpendicular to the surface, said luminous componentsincluding supercritical luminous components for which the angle θ isstrictly greater than a critical angle θ_(c)=arcsin(n_(L)/n_(s)), andalso including critical or subcritical luminous components for which theangle θ is less than or equal to the critical angle θ_(c), theobservation device being capable of: capturing at least part of thelight emission from a region of interest of the sample and obtaining acaptured luminous signal comprising luminous components which originatefrom the supercritical luminous components of the light emission;applying filters to the captured luminous signal in order to selectivelydecrease the amplitude and/or change the phase of some of the luminouscomponents of the captured luminous signal to obtain a filtered luminoussignal; and transforming the filtered luminous signal into an image zoneof the region of interest of the sample; the observation device beingfurther capable of: producing a modulation of the filtered luminoussignal, by allowing luminous components which originate from thecritical or subcritical luminous components of the light emission topass through, in order to obtain image zones of one and the same regionof interest of the sample, the modulation being applied to all or someof the luminous components of the captured luminous signal whichoriginate from the supercritical luminous components of the lightemission; and producing at least one useful image zone of the sample bycombining the image zones, so that the combination evidences differencesbetween the image zones, said differences being produced by themodulation.
 14. The observation device of claim 13, arranged so that theluminous components of the captured luminous signal that are concernedwith said modulation are originating from the supercritical luminouscomponents of the light emission.
 15. The observation device of claim13, arranged for applying the filters successively to the capturedluminous signal for obtaining successively the image zones which producethe useful image zone of the sample by combination of said image zones.16. The observation device of claim 13, arranged for capturing the imagezones simultaneously in order to obtain the useful image zone.
 17. Theobservation device of claim 16, arranged for splitting the capturedluminous signal, and for applying one of the filters to each splitcaptured luminous signal.
 18. The observation device of claim 13,comprising a plurality of filters such that: a filter of said pluralityof filters allows the passing through of, in the filtered luminoussignal, luminous components which originate from the supercriticalluminous components of the light emission; the filters of said pluralityof filters all allow the luminous components of the captured luminoussignal which originate from the critical or subcritical luminouscomponents of the light emission to pass through, and are effectivesubstantially in a same way onto the luminous components of the capturedluminous signal which originate from the critical or subcriticalluminous components of the light emission; and there are at least twofilters of said plurality of filters that are effective substantially indifferent ways onto the amplitude or the phase of at least some of theluminous components of the captured luminous signal which originate fromthe supercritical luminous components of the light emission; and theobservation device allowing to capture a plurality of image zones of oneand same region of interest of the sample using the plurality offilters, each filter being useful for capturing one image zone among theplurality of image zones, and allowing to produce the useful image zoneof the sample by a calculation combining the plurality of image zones soas to evidence the differences between the image zones of the pluralityof image zones of the sample.
 19. The observation device of claim 13,comprising two filters such that: one of the two filters allows thepassing through, in the filtered luminous signal, of the luminouscomponents which originate from the supercritical luminous components ofthe light emission; and the other filter is effective substantially inthe same way as said one of the two filters onto the luminous componentsof the captured luminous signal which originate from the critical orsubcritical luminous components of the light emission, and decreasessubstantially more than said one of the two filters the amplitude of atleast some of the luminous components of the captured luminous signalwhich originate from the supercritical luminous components of the lightemission; and the observation device allowing to capture at least twoimage zones of one and same region of interest of the sample using bothfilters, each filter being useful for capturing one of the image zones,and allowing to produce the useful image zone of the sample by acalculation combining both image zones of the sample, wherein thecalculation comprises an algebraic difference between both image zonesof the sample.
 20. The observation device of claim 13, wherein thefilters also partially reduce the amplitude of all or some of theluminous components of the captured luminous signal which originate fromthe critical and subcritical luminous components of the light emission.21. The observation device of claim 13, wherein luminous components ofthe captured luminous signal which originate from luminous components ofthe light emission that form one and same value for the angle θ, areprocessed substantially in a same way by one and same filter fordecreasing the amplitude or changing the phase.
 22. The observationdevice of claim 13, comprising: a neutral filter that allows to passthrough, in the filtered luminous signal and without any decrease ofamplitude, all the luminous components of the captured luminous signalwhich originate from the supercritical luminous components of the lightemission, for providing one of the image zones of the sample; and atotal filter which cancels in the filtered luminous signal all theluminous components which originate from the supercritical luminouscomponents of the light emission, for providing another one of the imagezones.
 23. The observation device of claim 13, comprising a full-fieldimmersion lens and the filters are located in a rear focal plane of theimmersion lens and/or in a conjugate plane of said rear focal plane. 24.The observation device of claim 23, comprising a diaphragm that allows,in an open position, the passing through of luminous components of thecaptured luminous signal which originate from the supercritical luminouscomponents of the light emission and allows, depending on the degree ofclosure of the diaphragm, to obscure the luminous components of thecaptured luminous signal which originate from the supercritical luminouscomponents of the light emission having an angle θ greater than a limitvalue related to said degree of closure.